(1) Field of the invention
The present invention generally relates to tissue engineering. More specifically, the invention is directed to improved tissue scaffolds that comprise transgenic bioactive molecules.
(2) Description of the Related Art
Tissue engineering using scaffolds seeded with vertebrate cells are widely used to produce varied tissues that are utilized in vivo. Scaffolds can be used to make numerous tissue types including vascular tissue (US Patent Application Publication 2003/0068817), cardiac or skeletal muscle, nervous tissue (US Patent Application Publication 2004/0078090), skin (WO99/43787), and others (See, e.g., US Patent Application Publications 2004/0175366, 2004/0078090 and US 2003/0068817, and PCT Publications WO99/43787, WO03/041568, WO03/044164, and WO03/082145). Scaffolds for various connective tissues (e.g., bone, cartilage, tendon, meniscus, fibrocartilage and ligament) are particularly well developed (Sharma and Elisseeff, 2004; Frenkel and DiCesare, 2004; Almarza and Athanasiou, 2004; U.S. Pat. Nos. 6,737,053; 6,214,369; 6,398,816; 5,906,934; 5,700,289; 4,846,835; PCT Publication WO03/043486).
Further development of tissue engineering methods is particularly needed for rotator cuff tears. Rotator cuff tears are a very common cause of pain and disability. The tendon stabilizes the shoulder by holding the head of the humerus in the glenoid cavity of the scapula (Dahlgren et al., 2001). Occupational shoulder injuries are one of the most frequent patient complaints, second only to back pain (Soslowsky et al., 1997). Among the tendons of the rotator cuff of the shoulder, the supraspinatus tendon is the one often most affected (Thomopoulos et al., 2002). Even though the incidence of rotator cuff tear and shoulder injury is high, the pathophysiology of rotator cuff injury and healing is poorly understood.
Tendon architecture consists of collagen fibrils embedded in a matrix of proteoglycan (Wada et al., 2001). Fibroblasts are the predominant cell type within tendons, and they are arranged in spaces between the parallel collagen bundles (Soslowsky et al., 1997; Abboud and Soslowsky, 2002). The major constituent of tendon is type 1 collagen (Wada et al., 2001). During the first week of tendon healing, proliferating tissue from the paratenon penetrates the gap between the tendon stumps and fills it with undifferentiated and disorganized fibroblasts. Capillary buds invade the area and together with the fibroblasts compose the granulation tissue between the tendon ends. Collagen synthesis can be detected by the third day. After about two weeks, the tendon stumps appear to be fused by a fibrous bridge. Dramatic fibroblast proliferation and collagen production in the granulation tissue continue. Between the third and fourth weeks, fibroblasts and collagen fibers near the tendon begin to orient themselves along the long axis Of the tendon as a result of stress. Only collagen near the tendon reorganizes, the more distant scar-like tissue remains unorganized (Wada et al., 2001).
To date, there is no ideal treatment method for rotator cuff healing. The endogenous healing is poor or insufficient in most rotator cuff tears and especially in large massive tears (Carpenter et al., 1998; Skutek et al., 2001). The current technique of suturing the parts of the tendon together does not give the desired improvement in outcome (Skutek et al., 2001). Ideally the best strategy would be one that potentiates endogenous healing processes. Recently, there have been many studies dealing with the properties of the rotator cuff tendon and its intrinsic healing properties (Kobayashi et al., 2001; Thomopoulos et al., 2002; Choi et al., 2002; Premdas et al., 2001; Desmouliere et al., 1993; Nakase et al., 2002; Aspenburg and Forslund, 1999). Kobayashi et al. showed that in the healing of full-thickness tears of avian supracoracoid tendon, the expression of α1(III) lasted longer than α1(I) procollagen mRNA (Kobayashi et al., 2001). Additionally, the healing process progresses from the bursal side to the joint side (Kobayashi et al., 2001; Premdas et al., 2001). This was shown in an experimentally created full thickness tear of rotator cuff tendon (Kobayashi et al., 2001). In a rat model study, it was noted that type XII collagen, aggrecan and biglycan was also increased in the healing tissue (Thomopoulos et al., 2002). Type XII collagen is fibril-associated collagen that binds to type I collagen and projects into ground matrix (Thomopoulos et al., 2002). In an acute supraspinatus tendon model tear in rabbits, it was shown that matrix metalloprotinease (MMP-2), a collagen degrading enzyme, may inhibit the healing process (Choi et al., 2002). Tissue inhibitor matrix metalloprotinease 1 (TIMP-I), the inhibitor of the MMP family, seems to enhance healing suggesting that inadequate TIMP-I is responsible for poor healing (Choi et al., 2002). However, the most interesting finding was that a large percentage of fibroblasts in the torn human rotator cuff contain smooth muscle actin (SMA) (Premdas et al., 2001). TGF-β1 increases the level of SMA, and thereby fibroblasts, in these tissues. Myofibroblasts have been proposed to play a role in wound contracture and retractile phenomenon observed during the fibrotic process (Desmouliere et al., 1993; Abboud and Soslowsky, 2002). Cartilage-derived morphogenic protein 1 (CDMP 1), a member of the bone morphogenic protein (BMP) superfamily has been identified as having chondrogenic activity (Nakase et al., 2002; Aspenburg and Forslund, 1999). In addition, GDF-5, the mouse homologue of CDMP-I reportedly induces the development of tendon tissue in vivo when implanted ectopically and enhances tendon healing in rats (Nakase et al., 2002; Aspenburg and Forslund, 1999). The pattern of localization of GDF-5 was found to be similar to that of collagen 1 (Col 1) messenger RNA (mRNA) Nakase et al., 2002; Aspenburg and Forslund, 1999). In light of these studies, it is possible that the combination of GDF-5 and TGF-β may play a crucial role in the healing process. Theoretically, GDF-5 will induce the development of tendon tissue by specifically directing the synthesis of collagen. TGF-β, meanwhile, will induce contractile activity and thereby, proliferate the reparative process.
Surgically demonstrable full thickness tears are present in about ⅕th of all elderly patients. The etiology of the tear includes impingement syndrome, instability, trauma, etc. (Malickey et al., 2002; Lewis et al., 2001). Impingement syndrome, which constitutes 75% of rotator cuff etiology, describes pain in the subacromial space when the humerus is elevated or internally rotated (Malickey et al., 2002; Rickert et al., 2001). During humeral flexion, the supraspinatus tendon and bursa become entrapped between the antero inferior corner of the acromion (and coraco-acromial ligament) and the greater tuberosity (Carpenter et al., 1998). This syndrome is thought to precipitate attritional changes in the rotator cuff, leading to rotator cuff tear (Carpenter et al, 1998; Bey et al., 2002). Once the supraspinatus (and infraspinatus) tendon is disrupted there will often be further impingement and irritation, which can lead to biceps tendonitis and subsequent rupture (Carpenter et al., 1998). Shoulder instability could be anterior or multidirectional (Carpenter et al., 1998). In either case, it happens to young active athletes that are involved overhead throwing sports. Non-operative treatment for minor rotator cuff tears includes rest and gentle stretch and strengthening (Jann et al., 1999). The current gold standard for rotator cuff tear, however, is surgical repair of tendon (Carpenter et al., 1998). Animal models have been developed for both tendon and ligament tissue engineering (Carpenter et al., 1999).
Cells seeded onto scaffolds generally do not produce sufficient cytokines to provide for optimum growth and colonization of the scaffold, improved colonization of the scaffold can be achieved by utilizing cells that are genetically engineered to produce increased cytokine levels. See, e.g., U.S. Pat. No. 6,398,816. However, transplantation of scaffolds comprising viable transgenic cells can lead to problems such as excessive production of the transgenic cytokines and/or spread and growth of the transgenic cells away from the transplanted area.
Thus, there is a need for improved methods for utilizing transgenic cells on scaffolds for tissue engineering. The present invention addresses that need.